Modulation of O-linked N-acetylglucosamine levels on nuclear and cytoplasmic proteins in vivo using the peptide O-GlcNAc-beta-N-acetylglucosaminidase inhibitor O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate.
O-Linked N-acetylglucosamine (O-GlcNAc) is a ubiquitous and abundant post-translational modification found on nuclear and cytoplasmic proteins and is thought to be a dynamically regulated modification much like phosphorylation. In this study we have demonstrated that O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbama te (PUGNAc), a potent in vitro inhibitor of the enzyme responsible for the removal of O-GlcNAc from proteins (peptide O-GlcNAc-beta-N-acetylglucosaminidase), can be used to increase O-GlcNAc levels on nuclear and cytoplasmic proteins in vivo. Overall, PUGNAc caused approximately a 2-fold increase in O-GlcNAc levels in the human colon cancer cells, HT29, although the effects on individual proteins varied. The increase appeared to be the result of the direct inhibition of the peptide O-GlcNAc-beta-N-acetylglucosaminidase since neither the O-GlcNAc transferase nor UDP-GlcNAc levels were affected by the treatment. O-GlcNAc levels in other cell lines tested (NIH 3T3, CV-1, and HeLa) were also affected by PUGNAc, although the effects on HeLa cells were minimal. At the concentrations tested, PUGNAc was non-toxic and had no affect on the growth rate of any of the cell lines examined. Interestingly, we demonstrated that an increase in O-GlcNAc levels on the transcription factor Sp1 resulted in a reciprocal decrease in its level of phosphorylation, supporting the hypothesis that O-GlcNAc competes with phosphate on some proteins. These studies demonstrate that PUGNAc is an effective inhibitor of O-GlcNAc turnover within cells and can be used to selectively alter the extent of O-GlcNAc on cellular proteins.
Haltiwanger
R S
RS
Department of Biochemistry and Cell Biology, Institute for Cell and Developmental Biology, State University of New York, Stony Brook, New York 11794-5215, USA.
Grove
K
K
Philipsberg
G A
GA
D000117:Acetylglucosamine / Q000031:analogs & derivatives* / Q000378:metabolism* / Q000494:pharmacology; D000118:Acetylglucosaminidase / Q000037:antagonists & inhibitors*; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006031:Glycosylation; D006367:HeLa Cells; D051548:Histone Acetyltransferases; D006801:Humans; D009097:Multienzyme Complexes; D010091:Oximes / Q000494:pharmacology*; D048448:Phenylcarbamates; D001619:beta-N-Acetylhexosaminidases
D004791:Enzyme Inhibitors; D009097:Multienzyme Complexes; D010091:Oximes; D048448:Phenylcarbamates; C068836:N-acetylglucosaminono-1,5-lactone O-(phenylcarbamoyl)oxime; D051548:Histone Acetyltransferases; C110628:hexosaminidase C; D000118:Acetylglucosaminidase; D001619:beta-N-Acetylhexosaminidases; D000117:Acetylglucosamine